Rejuvenating agent and rejuvenating method

Abstract

Provided is a rejuvenating agent that restores cell function, suppresses senescence-associated secretory phenotypes (SASP), extends telomeres, and restores cell division capability.　This rejuvenation agent is for use by administration to subjects having cells and contains microparticles derived from dental-pulp-derived stem cells. The present invention also provides a rejuvenation method.

Description

Rejuvenating agents and rejuvenating methods

[0001] The present invention relates to a rejuvenation agent and a rejuvenation method.

[0002] There have been many studies on the rejuvenation of cells and tissues, as well as the organisms that contain them. In previous studies, rejuvenation methods have been considered to involve at least one of the following: restoring cellular function, suppressing senescence-associated secretory phenomena (SASP), lengthening telomeres, and restoring cell division ability.

[0003] Regarding the restoration of cell function, Patent Document 1 describes a cell-free supernatant or a fraction of the cell-free supernatant that has previously supported the growth of a dedifferentiated plant cell suspension culture, for use as a drug for skin treatment, the cell-free supernatant or fraction containing a peptide of 4 to 300 amino acids selected from peptide plant growth factors, plant transcription factors, epigenetic factors, and mixtures thereof, and the cell-free supernatant or fraction is free of cytoplasmic intracellular contents resulting from cell lysis and membranes and / or cell walls. It also describes the use of the cell-free supernatant or fraction as a repair and / or regenerator for animal skin cells by reversing aging in skin cells (i.e., fibroblasts).

[0004] Regarding the inhibition of SASP, Patent Document 2 describes a method for extending the lifespan of a subject in need thereof, comprising administering therapeutically effective amounts of berberine, a vitamin A compound, and α-ketoglutaric acid (AKG) to the subject. It also describes that SASP secreted by senescent cells contributes to the onset and progression of aging-related diseases. Non-Patent Document 1 describes that treatment of aged animals with small extracellular vesicles (sEVs) derived from adipose mesenchymal stem cells of young animals resulted in improvements in motor coordination, grip strength, fatigue resistance, fur regeneration, renal function, and the like, as well as a reduction in aging markers of SASP (IL-1β and IL-6).

[0005] Telomeres are repetitive DNA sequences that cap the ends of each chromosome and are accompanied by proteins that ensure and guarantee chromosomal integrity by protecting the ends of each chromosome from constant degradation during each cell cycle. Telomere shortening can lead to cellular aging, and telomeres gradually shorten during normal aging. Increasing the length of telomeres in cells leads to cellular rejuvenation. In contrast, a method for rejuvenating cells by contacting them with a specific agent to lengthen telomeres is known. For example, Patent Document 3 describes a method for increasing telomere length in one or more human cells, which includes contacting one or more human cells with an agent that increases Zscan4 expression in the human cells, whereby increased Zscan4 expression induces telomere length in the one or more human cells compared to one or more corresponding human cells not contacted with the agent.

[0006] Regarding the restoration of cell division ability, the cell division ability generally declines with age. In contrast, by restoring the cell division ability, new healthy cells are generated.

[0007] JP 2018-512440 A JP 2023-027149 A JP 2023-099826 A

[0008] Sci. Adv. (2022) 8, 42, eabq2226

[0009] However, until now, there have been few known rejuvenating agents or methods that can restore cell function, suppress senescence-associated secretory phenomena (SASP), lengthen telomeres, and restore cell division ability.

[0010] The problem to be solved by the present invention is to provide a rejuvenating agent that has all of the following effects: recovery of cell function, suppression of secretory phenomena associated with cell aging, lengthening of telomeres, and recovery of cell division ability.

[0011] The inventors have discovered that a composition comprising a culture supernatant of dental pulp-derived stem cells, which contains microparticles derived from dental pulp-derived stem cells, restores cell function, suppresses secretory phenomena associated with cellular senescence, lengthens telomeres, and restores cell division ability.

[0012] Specifically, the present invention and preferred configurations thereof are as follows.

[0013] [1] A rejuvenating agent for administration to a subject containing cells, the rejuvenating agent comprising microparticles derived from dental pulp-derived stem cells. [2] The rejuvenating agent according to [1], a composition comprising a culture supernatant of dental pulp-derived stem cells, the culture supernatant comprising microparticles. [3] The rejuvenating agent according to [1], wherein the microparticles comprise (1) at least one of the following rejuvenation-related miRNAs: Rejuvenation-related miRNAs: has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has-miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p, and miR-93-5p. [4] The rejuvenation agent according to [3], wherein the microparticles contain at least one of has-miR-125a-5p, hsa-miR-199a-3p, and hsa-miR-21-5p as the rejuvenation-related miRNA. [5] The rejuvenating agent according to [3], wherein the microparticles contain rejuvenation-related miRNA at a higher concentration than in the culture supernatant. [6] The rejuvenating agent according to [1], wherein the composition does not contain a culture supernatant excluding microparticles. [7] (2) The rejuvenating agent according to [1], which is an inhibitor of the expression of the following SASP factors: IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3, and MCP-1. [8] The rejuvenating agent according to [1], wherein the cells are somatic stem cells or differentiated cells. [9] The rejuvenating agent according to [1], wherein the subject is a somatic stem cell or a differentiated cell, and administration of the rejuvenating agent to a subject that has been passaged n times, where n is a natural number, can rejuvenate the subject more effectively than a subject that has been passaged n times without administration of the rejuvenating agent.

[10] (3) The rejuvenating agent according to [1], which suppresses shortening of the average telomere length at the end of chromosomes possessed by cells.

[11] (4) The rejuvenating agent according to [1], which suppresses the number of cells positive for SA-β-gal staining when administered to a subject that has been passaged n times, compared to the number of cells positive for SA-β-gal staining in a subject that has been passaged n times without administration of the rejuvenating agent.

[12] The rejuvenating agent according to [1], which has all of the effects of restoring cell function, suppressing senescence-associated secretory phenomenon (SASP), lengthening telomeres, and restoring cell division ability.

[13] The rejuvenating agent according to [1], which satisfies all of the following (1) to (4): (1) Rejuvenation-related miRNAs including at least one of the following rejuvenation-related miRNAs: has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has-miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p, and miR-93-5p; (2) An inhibitor of the expression of the following SASP factors: SASP factors: IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3, and MCP-1; (3) Suppressing the shortening of the average telomere length at the end of chromosomes in cells; (4) Suppressing the number of cells that are positive for SA-β-gal staining in a subject that has been passaged n times when the rejuvenating agent is administered compared to the number of cells that are positive for SA-β-gal staining in a subject that has been passaged n times without administration of the rejuvenating agent.

[14] The rejuvenating agent according to [1], wherein the microparticles are exosomes.

[15] A rejuvenation method comprising administering an effective amount of the rejuvenating agent according to [1] to a subject containing cells.

[16] The rejuvenation method according to

[15] , wherein the amount of the rejuvenating agent administered is an amount that results in 500 or more microparticles per cell.

[0014] According to the present invention, it is possible to provide a rejuvenating agent that has all of the following effects: recovery of cell function, suppression of senescence-associated secretory phenomenon (SASP), lengthening of telomeres, and recovery of cell division ability.

[0015] Figure 1 is a graph showing the read counts of 50 miRNAs with the highest read counts among miRNAs expressed in exosomes from dental pulp-derived stem cells. Figure 2 is a graph showing the expression of each SASP factor as a relative value (%), with Untreated set to 100. Figure 3 is a graph showing the average telomere length of each chromosome end of NHDF cells at passage number P5 and the average telomere length of each chromosome end of NHDF cells at passage number P12 when stimulated with the rejuvenating agent of Example 1 (culture supernatant of dental pulp-derived stem cells) or control (phosphate-buffered saline; PBS). Figure 4 is a graph showing the percentage of SA-β-gal-positive cells in senescent ADMSCs (adipose-derived stem cells) at passage number P12 when stimulated with the rejuvenating agent of Example 1 (culture supernatant of dental pulp-derived stem cells) or control (phosphate-buffered saline; PBS) for 72 hours (Day 3). Figure 5 is a schematic diagram of Test Example 4 showing that the rejuvenating agent of the present invention (SHED-CM-EVs) has the effect of rejuvenating aged mesenchymal stem cells. Figure 6 is a graph showing the relationship between the passage number of ADMSCs at 0 hours of culture time and the number of SA-β-gal positive cells.

[0016] The present invention will be described in detail below. The following description of the constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0017] [Rejuvenating Agent] The rejuvenating agent of the present invention is a rejuvenating agent for administration to a subject containing cells, and contains microparticles derived from dental pulp-derived stem cells. The rejuvenating agent of the present invention has all of the following effects: restoring cell function, suppressing senescence-associated secretory phenomenon (SASP), lengthening telomeres, and restoring cell division ability. Preferred embodiments of the rejuvenating agent of the present invention are described below.

[0018] <Definition of rejuvenation> In the present invention, rejuvenation refers to the restoration of cell function, the suppression of senescence-associated secretory phenomena, the lengthening of telomeres, and the restoration of cell division ability. Therefore, it is preferable that the rejuvenating agent of the present invention restores cell function, the suppression of senescence-associated secretory phenomena (SASP), the lengthening of telomeres, and the restoration of cell division ability.

[0019] Specifically, the rejuvenating agent of the present invention preferably satisfies at least one of the following (1) to (4), more preferably at least two, particularly preferably at least three, and most particularly preferably all: (1) is related to the recovery of cellular function, (2) is related to the suppression of senescence-associated secretory phenomenon (SASP), (3) is related to the lengthening of telomeres, and (4) is related to the recovery of cell division ability. (1) Rejuvenation-related miRNAs including at least one of the following rejuvenation-related miRNAs: has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has-miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p, and miR-93-5p; (2) An inhibitor of the expression of the following SASP factors: SASP factors: IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3, and MCP-1; (3) Suppressing the shortening of the average telomere length at the end of chromosomes possessed by cells; (4) Suppressing the number of positive cells stained with SA-β-gal contained in a subject that has been passaged n times when a rejuvenating agent is administered compared to the number of positive cells stained with SA-β-gal contained in a subject that has been passaged n times without administration of a rejuvenating agent.

[0020] <Culture Supernatant of Dental Pulp-Derived Stem Cells> The rejuvenating agent of the present invention is preferably a composition containing a culture supernatant of dental pulp-derived stem cells, and in this case, the culture supernatant preferably contains microparticles.

[0021] <Microparticles> The rejuvenating agent of the present invention contains microparticles derived from dental pulp-derived stem cells. The microparticles are derived from dental pulp-derived stem cells, for example, by secretion, budding, or dispersion from mesenchymal stem cells of dental pulp-derived stem cells, and are exuded, released, or shed into a cell culture medium. The origin of the microparticles can be determined using known methods. For example, the method described in J Stem Cell Res Ther (2018) 8:2 can be used to determine whether the microparticles are derived from dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord-derived stem cells, or other stem cells. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticles.

[0022] (1) Rejuvenation-related miRNA In the present invention, the microparticles preferably contain at least one of the following rejuvenation-related miRNAs: has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has-miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p, and miR-93-5p. In the present invention, miRNA (MicroRNAs) are RNA molecules of, for example, 21 to 25 bases (nucleotides). miRNA can regulate gene expression by degrading target mRNA or inhibiting its decoding. In the present invention, miRNA may be, for example, single-stranded (monomer) or double-stranded (dimer). Furthermore, in the present invention, miRNA is preferably mature miRNA cleaved by ribonuclease such as Dicer.

[0023] The sequences of miRNAs described herein, such as hsa-miR-199a-3p, are registered in publicly known databases (e.g., the miRBase database) with associated accession numbers, allowing those skilled in the art to unambiguously determine the sequences. For example, the accession number for hsa-miR-199a-3p is MIMAT0000232, and the sequence is registered in the miRBase database. Hereinafter, the accession number for each miRNA will be omitted. However, the miRNAs referred to herein also include variants that differ by approximately 1 to 5 bases from mature miRNAs such as hsa-miR-199a-3p. Furthermore, each miRNA referred to herein includes a polynucleotide consisting of a base sequence identical to the base sequence of each miRNA (e.g., hsa-miR-199a-3p), or a polynucleotide consisting of a complementary base sequence thereof, and which has the function of the miRNA of the present invention. "Identity" refers to the degree of identity when the sequences being compared are appropriately aligned, and refers to the percentage of exact amino acid matches between the sequences. Alignment can be performed using any algorithm, such as BLAST. The identity is, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or approximately 99%. A polynucleotide consisting of an identical nucleotide sequence may have, for example, point mutations, deletions, and / or additions in the nucleotide sequence of an miRNA. The number of nucleotides such as point mutations is, for example, 1 to 5, 1 to 3, 1 to 2, or 1. Furthermore, a polynucleotide consisting of a complementary nucleotide sequence is, for example, a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of an miRNA nucleotide sequence and includes a polynucleotide having the function of the miRNA of the present invention. Stringent conditions are not particularly limited, but include, for example, the conditions described in

[0028] of JP 2017-184642 A, the contents of which are incorporated herein by reference.

[0024] In the present invention, the microparticles preferably contain rejuvenation-related miRNA (and TERT-related miRNA, described below) at a higher concentration than that of the culture supernatant of dental pulp-derived stem cells. Preferred embodiments of the rejuvenation-related miRNA contained in the microparticles are described below.

[0025] Human miR-125a and miR-125b correspond to lin-4 in C. elegans, and it is known that lin-4 directly regulates lifespan in C. elegans, with loss of lin-4 function shortening lifespan (Human Genetics (2020) 139, 291-308). Therefore, human miR-125a-5p and has-miR-125b-5p are speculated to be miRNAs associated with lifespan. miR-125b is known to be transferred from young to old fibroblasts via exosomes, promoting fibroblast migration and migration, thereby combating aging (Journal of Nanobiotechnology (2020), 20, 144). Therefore, has-miR-125b-5p can be considered a rejuvenation-related miRNA for anti-aging.

[0026] Int. J. Mol. Sci. (2020), 21(15):5281, Fig. 2, provides a schematic diagram showing the role of microRNA regulation in human skin, including the involvement of miRNAs in the development of skin components, the aging process, and cancer. This diagram shows that miR-199a and miR-199b are involved in hair development, miR-21 and miR-155 are involved in the development of Langerhans cells, and miR-155 is involved in photoaging of fibroblasts. Therefore, miR-155-5p, miR-199a-3p, miR-199b-5p, and miR-21-5p can be considered rejuvenation-related miRNAs. Also, in Int. J. Mol. Sci. (2020), 21(15):5281, Fig. Figure 3 presents a schematic diagram of the role of microRNAs in regulating key molecular signaling pathways in the skin aging process. This diagram shows miRNAs (shown in red) that regulate cellular signaling mechanisms and potentially promote aging, as well as miRNAs (shown in blue) that suppress aging. It also shows that miR-93 is involved in the inhibition of MMPs, P21, TGFβ, and E2F, miR-24 is involved in the inhibition of P16, miR-155 is involved in the inhibition of c-JUN, and miR-181a / b is involved in the inhibition of SIRT1. Therefore, miR-155-5p, miR-181a-5p, miR-24-3p, and miR-93-5p can be considered rejuvenation-related miRNAs.

[0027] Communications Biology (2021), vol. 4, p. 427, reported that miR-199-3p, a cell-free miRNA circulating in the blood, was significantly reduced in the blood of aging mice compared to young mice. Furthermore, it was shown that miR-199-3p promotes muscle differentiation and regeneration. Administration of a miR-199 mimic that supplies miR-199-3p to aging mice delayed muscle fiber hypertrophy and muscle strength loss. Systemic administration of a miR-199 mimic to mdx mice, an animal model of Duchenne muscular dystrophy (DMD), significantly improved muscle strength. Therefore, cell-free miR-199a-3p and miR-199b-3p in the blood can be considered rejuvenation-related miRNAs that exert anti-aging effects, such as hypertrophy of aging muscle fibers. ACS Nano (2019) 13, 11273-11282 reported that hsa-miR-196a-5p and hsa-miR-744-5p were downregulated in exosomes from three-dimensional spheroid-formed human fibroblasts (HDFs) compared to exosomes from monolayer-cultured human fibroblasts (2D HDFs), whereas hsa-miR-133a-3p, hsa-miR-223-3p, hsa-5011-5p, hsa-miR-325, hsa-miR-199b-5p, and hsa-miR-34a-5p were upregulated in exosomes from mesenchymal stem cells (MSCs) and 2D HDFs (Figure 2B). It has also been reported that UV rays induce increased oxidative stress in the skin and activate the TNF-α and NF-kB signaling pathways, resulting in collagen degradation and aging. 3D HDF exosomes have been reported to upregulate TIMP-1 and TGF-β, which are important for suppressing MMPs and regulating matrix synthesis, while TNF-α is downregulated.It has been reported that during the aging process, upregulation of MMP production and downregulation of collagen production lead to age-related skin disorders such as weakening of the dermal structure and reduced functional integrity. It has been shown that exosomes from 3D spheroidized HDFs are more effective in restoring the function of aged HDFs than exosomes from mesenchymal stem cells (MSCs) or 2D HDFs. It has also been reported that exosomes from spheroidized HDFs can regulate dermal fibroblasts, induce efficient collagen biosynthesis, and potentially ameliorate skin inflammation induced by UVB irradiation. It has been suggested that upregulation of miR-133a and miR-223 may contribute to this improvement. Furthermore, it has been reported that exosomes from spheroid-formed HDFs suppress UVB-induced MMP1 expression, restore type I procollagen, and activate the TGF-β signaling pathway, and that exosomes from spheroid-formed HDFs improve skin inflammation and aging through downregulation of TNF-α. Therefore, the above miRNAs, especially miR-223-3p, can be said to be rejuvenation-related miRNAs that improve age-related skin disorders.

[0028] In the present invention, it is preferable that the microparticles contain at least one of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p as rejuvenation-related miRNAs, more preferably at least two of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p, and most preferably all of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p.

[0029] The microparticles preferably contain at least one type of rejuvenation-related miRNA, with a read count obtained by analysis using IMOTA of 10,000 or more, more preferably 30,000 or more, particularly preferably 50,000 or more, and more preferably 100,000 or more. The microparticles preferably contain has-miR-125a-5p and hsa-miR-199a-3p, with a Log2 Ratio of the read count obtained by analysis using IMOTA of 30,000 or more, particularly preferably 50,000 or more, and more preferably 100,000 or more. The microparticles preferably contain hsa-miR-21-5p at 10,000 or more, more preferably 30,000 or more. The microparticles preferably contain at least one type of rejuvenation-related miRNA, as a Log2Ratio of the read count number obtained by analysis using IMOTA, of 8.0 or more, more preferably 10.0 or more, and particularly preferably 12.0 or more. The microparticles preferably contain has-miR-125a-5p, hsa-miR-199a-3p, and hsa-miR-21-5p, as a Log2Ratio of the read count number obtained by analysis using IMOTA, of 8.0 or more, more preferably 10.0 or more, and particularly preferably 12.0 or more.

[0030] The microparticles of the present invention preferably have expression levels of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p that are 1.1 times or more, more preferably 1.5 times or more, and particularly preferably 2 times or more, compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells.

[0031] Here, microparticles derived from the culture supernatant of dental pulp-derived stem cells contain approximately 2,600 types of small RNAs. Of these, approximately 1,800 types are miRNAs. Of these miRNAs, 180 to 200 types are highly abundant. The miRNAs highly abundant in microparticles derived from dental pulp-derived stem cells are characterized by their high content of microRNAs related to the treatment of cranial nerve diseases and rejuvenation-related miRNAs, a previously unknown finding newly discovered by the present inventors. This characteristic is significantly different from the types of miRNAs highly abundant in other mesenchymal stem cell microparticles. For example, the miRNAs highly abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to the treatment of cranial nerve diseases. In the context of the present invention, the rejuvenation-related miRNAs in microparticles derived from dental pulp-derived stem cells are significantly more abundant than the rejuvenation-related miRNAs in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells.

[0032] The microparticles preferably contain two or more types of rejuvenation-related miRNA, more preferably three or more types, even more preferably five or more types, particularly preferably eight or more types, and even more particularly preferably ten or more types.

[0033] (2) SASP Factor Expression Inhibitor The rejuvenating agent of the present invention is preferably an expression inhibitor of the following SASP factors. SASP factors: IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3, and MCP-1. The rejuvenating agent of the present invention can slow the aging of cells (such as stem cells or differentiated cells, particularly tissue stem cells) and reduce the SASP factors, which are aging factors released by senescent cells (particularly senescent stem cells). Of these SASP factors, the rejuvenating agent of the present invention is more preferably an expression inhibitor of CCL5, CXCL10, IL-6, TNF-α, and MCP-1. Furthermore, the active ingredient as an expression inhibitor of SASP factors in the rejuvenating agent of the present invention is not particularly limited, but it is preferable that the microparticles contained in the rejuvenating agent of the present invention are expression inhibitors of these SASP factors.

[0034] IL-1β is a potent inflammatory cytokine that lowers pain threshold, induces fever, and causes tissue injury. It promotes T cell activation and antigen recognition, induces Th17 differentiation and maturation, and is involved in the induction and secretion of prostaglandins. IL-1β mRNA transcription is induced by microbial components, TNFα, IL-18, IL-1α, and IL-1β. CCL5 (Chemokine (C-C motif) ligand 5) and CXCL10 (C-X-C motif chemokine 10) are chemokines that attract cytotoxic T cells that kill cancer cells. PAI-1 (plasminogen activator inhibitor-1) is present in vascular endothelial cells, the liver, platelets, and adipocytes, and is released in large amounts into the blood upon vascular endothelial damage or platelet destruction. PAI-1 regulates fibrinolytic responses by specifically and immediately inhibiting tissue plasminogen activator (t-PA), which aids in the production of plasmin, which dissolves formed thrombi. IL-6 is a B-cell differentiation factor; B-cell stimulating factor-2; hepatocyte-stimulating factor; hybridoma growth factor; and plasmacytoma growth factor. IL-6 is a representative inflammatory cytokine and a multifunctional cytokine involved in the control of acute inflammatory responses, regulation of specific immune responses including B- and T-cell differentiation, bone metabolism, platelet production, epithelial proliferation, menstruation, neuronal differentiation, neuroprotection, aging, cancer, and inflammatory responses occurring in Alzheimer's disease. IL-6 is thought to play a role in the development of numerous diseases and disorders, including fatigue, cachexia, autoimmune diseases, skeletal diseases, cancer, heart disease, obesity, diabetes, asthma, Alzheimer's disease, and multiple sclerosis (see paragraphs

[0002] to

[0005] of Japanese Patent Publication No. 2019-047787). Tumor necrosis factor (TNF) is a representative inflammatory cytokine. Known TNFs include TNFα, TNFβ, and LTβ. Among these, TNFα mediates several important life functions, including the structural and functional organization of secondary lymphoid organs, apoptosis and antitumor activity, inhibition of viral replication, immunoregulation, and inflammation.TNF also plays an important role in the pathogenesis of autoimmune diseases, acute phase responses, septic shock, fever, and cachexia (see

[0004] of JP 2020-079306 A). MMP-3 is a proteolytic enzyme produced by the lining cells of the synovial membrane of joints. It is closely associated with the pathology of rheumatoid arthritis (RA) by dissolving cartilage, and is therefore considered a marker reflecting synovial proliferation. The produced MMP-3 accumulates in synovial fluid, migrates into the blood via blood vessels and lymphatic vessels, and serum MMP-3 levels increase, thereby reflecting the degree of synovial proliferation in RA. MCP-1 (monocyte chemoattractant protein-1) is a chemokine belonging to the CC chemokine family and has a potent chemotactic effect on monocytes and macrophages. In arteriosclerosis, MCP-1 promotes macrophage infiltration into lesions, and progresses the pathology through mechanisms such as foam cell formation and secretion of various cytokines. In addition to monocyte and macrophage migration, MCP-1 also directly promotes the proliferation of vascular smooth muscle cells and collagen production by fibroblasts, and is involved in the pathology of various cardiovascular diseases.

[0035] (3) Telomere Lengthening / TERT-Related miRNA The rejuvenating agent of the present invention preferably extends the average telomere length at the chromosome ends of cells, or inhibits shortening of the average telomere length at the chromosome ends of cells. It is more preferable that the rejuvenating agent of the present invention inhibits shortening of the average telomere length at the chromosome ends of cells. The ability of the rejuvenating agent of the present invention to lengthen telomeres is demonstrated experimentally in the Examples below, and is also supported by the presence of TERT-related miRNA. Telomerase (TERT) confers replicative immortality to cancer cells, and its overexpression functions as a nearly universal marker of cancer. Because more than 50% of gene sequences encoding miRNAs are found in cancer-prone or highly mutated regions, miRNAs are involved in promoting cancer progression and silencing genes related to cancer progression. TERT-associated miRNAs have primarily been studied as oncogenic miRNAs, which activate TERT and elongate telomeres. Cancers (2020), 12(9), 2337, reported that miR-19b indirectly upregulates hTERT expression by inhibiting PITX1, an hTERT (human telomerase) suppressor gene. It also reported that miR-346 mediates hTERT upregulation through a competitive process with miR-138, which binds to the same region of the 3'UTR of hTERT mRNA as miR-346 but promotes the opposite effect on hTERT expression. In particular, it has been reported that miR-21 affects telomerase activity through downregulation of PTEN, thereby upregulating hTERT expression. Open J Proteom Genom (2016), 1(1): 013-018 describes that miR-21 inhibited apoptosis and induced proliferation accompanied by negative regulation of hTERT expression via the phosphoinositide 3-kinase (PI3K) signaling pathway by binding to the 3'UTR of PTEN, inhibiting its translation.Regarding miR-19b, it has been reported that PITX1 (Paired Like Homeodomain 1) mRNA is a direct target of miR-19b, and downregulation of PITX1 by miR-19b ultimately leads to enhanced expression of hTERT mRNA. The middle sequence motif (nt 8-13, CCGCAU) of miR-346 binds to G-rich sequence binding factor 1 (GRSF-1) to form a bulge loop. This binding has been reported to recruit hTERT mRNA to ribosomes for translation in an AG02-independent manner. It has been reported that miR-155 acts as an important regulatory factor in breast cancer cell development, efficiently increasing its expression with a decrease in TRF1 protein levels; miR-155 mediates telomere elongation, increased telomere fragility, and chromosome instability by reducing the expression of TRF1 (telomeric repeat factor 1), a part of the shelterin complex. PLoS ONE (2016), 11(9): e0162077 reports that when the expression levels of miRNAs expressed in the normal colonic mucosa of colon cancer patients and telomere length were observed, it was confirmed that high miRNA expression levels were associated with increased telomere length. Many miRNAs have been described as miRNAs that increase telomere length, such as miR-134-5p, miR-5088 (miR-5088-3p, miR-5088-5p), and miR-887 (miR-887-3p, miR-887-5p). PLoS ONE (2014), 9(4): e92088 describes combinations of miRNAs and TERT-related proteins that increase target genes, such as miR-181b and TERT, miR186 and TERF2IP, RAD50 and SIRT6, miR-96 and TERF2IP, and miR-15a and TATA box binding protein (TBP). For example, in a validation study of TERF2IP and RAD50, it was reported that only TERF2IP showed a significant decrease in transcript amount 60 minutes after exercise, accompanied by an increase in potentially binding miRNAs (miR-186 and miR-96).Based on this paper, miR-15a-5p, miR-181b-2-3p, miR-181b-3p, miR-181b-5p, miR-186-3p, miR-186-5p, and miR-96-5p can be considered TERT-related miRNAs. Almost all of these TERT-related miRNAs identified by IMOTA are contained in dental pulp-derived microparticles. In particular, six TERT-related miRNAs that target TERT are ranked within the top 50 miRNAs contained in dental pulp-derived stem cell microparticles: miR-16-5p, miR-143-3p, miR-181a-5p, miR-21-5p, miR-34a-5p, and miR-103a-3p. The active ingredient for telomere lengthening in the rejuvenation agent of the present invention is not particularly limited, but it is preferable that the microparticles contained in the rejuvenation agent of the present invention are the active ingredient for telomere lengthening. Although TERT-related miRNA promotes carcinogenesis, various miRNAs exist in the microparticles of dental pulp-derived stem cells and can regulate each other, so there is no problem of carcinogenicity (no need to conduct carcinogenicity experiments). TERT-related miRNA is also present in the body and is thought to be necessary for maintaining some kind of homeostasis.

[0036] (4) Restoration of Cell Division Ability The rejuvenating agent of the present invention is preferably capable of restoring cell division ability. Specifically, the rejuvenating agent of the present invention preferably suppresses the number of SA-β-gal-positive cells contained in a subject that has been passaged n times when the rejuvenating agent is administered, compared to the number of SA-β-gal-positive cells contained in a subject that has been passaged n times without administration of the rejuvenating agent. The number of SA-β-gal-positive cells (proportion of positive cells) is an index representing the cell division ability of cells and is expressed, for example, as a percentage. Generally, the number of SA-β-gal-positive cells (proportion of positive cells) decreases with the number of passages. n is an integer of 1 or greater. There is no limit to the value of n, but n may be, for example, 2 to 100, 4 to 20, 8 to 16, or 10 to 14. Regardless of the number of passages, administration of the rejuvenating agent of the present invention can rejuvenate the cells beyond the number of passages. The active ingredient in the rejuvenating agent of the present invention for restoring cell division ability is not particularly limited, but it is preferable that the microparticles contained in the rejuvenating agent of the present invention are the active ingredient for restoring cell division ability.

[0037] (Types of Microparticles) The microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, exovesicles, or microvesicles, and more preferably exosomes. The diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. Furthermore, it is desirable that tetraspanin molecules such as CD9, CD63, and CD81 are present on the surface of the microparticles, and this may be CD9 alone, CD63 alone, CD81 alone, or any combination of two or three of these. Below, preferred embodiments of using exosomes as microparticles will be described, but the microparticles used in the present invention are not limited to exosomes.

[0038] Exosomes are preferably extracellular vesicles released from cells upon fusion of multivesicular bodies with the plasma membrane. The surface of the exosomes preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells. The interior of the exosomes preferably contains intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins. Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another. Exosomes are easily traceable and can be targeted to specific regions.

[0039] (Microparticle Content) There is no particular limitation on the content of microparticles in the culture supernatant of dental pulp-derived stem cells. ８ It is preferable that the number of atoms contained is 1.0 × 10 or more. ８ It is more preferable that the number of atoms contained is 2.0 × 10 or more. ８ It is particularly preferable that the number of atoms contained is 2.5 × 10 or more. ８ It is more particularly preferable that the number of atoms contained is 1.0 × 10 or more. ９ It is even more particularly preferable that the culture supernatant of dental pulp-derived stem cells contains 1.0 x 10 microparticles or more. ８ It is preferable that the concentration is 2.0 × 10 ８ It is more preferable that the concentration is 4.0 × 10 ８ It is particularly preferable that the concentration is 5.0 × 10 ８ It is more particularly preferable that the content is 2.0 x 10 ９ In a preferred embodiment of the microparticles of the present invention, by containing such a large amount or high concentration of microparticles, the amount of rejuvenation-related miRNA used in the rejuvenation agent can be maintained high.

[0040] <Other Components> In addition to microparticles, the composition containing the culture supernatant of dental pulp-derived stem cells may contain other components depending on the type and purpose of administration to the animal to which it is administered, as long as the effects of the present invention are not impaired. Examples of other components include nutritional components, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, and antiseptics. Examples of nutritional components include fatty acids and vitamins. Examples of antibiotics include penicillin, streptomycin, and gentamicin. Examples of carriers include materials known as pharmaceutically acceptable carriers. The composition containing the culture supernatant of dental pulp-derived stem cells may be the culture supernatant of dental pulp-derived stem cells itself, or it may be a pharmaceutical composition further containing pharmaceutically acceptable carriers, excipients, and the like. The purpose of the pharmaceutical composition is to facilitate the administration of microparticles to the recipient.

[0041] The pharmaceutically acceptable carrier is preferably a carrier (including a diluent) that does not cause significant irritation to the subject to administration and does not abrogate the biological activity and properties of the administered compound. Examples of carriers include propylene glycol, (physiological) saline, emulsions, buffer solutions, culture media such as DMEM or RPMI, and cryopreservation media containing components that scavenge free radicals.

[0042] The composition containing the culture supernatant of dental pulp-derived stem cells may contain active ingredients of conventionally known rejuvenating agents. Those skilled in the art can appropriately modify the composition depending on the intended use, the subject of administration, etc.

[0043] On the other hand, compositions containing the culture supernatant of dental pulp-derived stem cells and the rejuvenation agent of the present invention preferably do not contain specific substances. Below, a composition containing the culture supernatant of dental pulp-derived stem cells will be described, but the same applies to the rejuvenation agent of the present invention. For example, a composition containing the culture supernatant of dental pulp-derived stem cells preferably does not contain dental pulp-derived stem cells. Furthermore, a composition containing the culture supernatant of dental pulp-derived stem cells preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. Examples of other cytokines include those described in paragraphs

[0014] to

[0020] of JP 2018-023343 A. Furthermore, a composition containing the culture supernatant of dental pulp-derived stem cells preferably does not contain Siglec-9. However, it may contain a sialic acid-binding immunoglobulin-like lectin other than Siglec-9. Furthermore, a composition containing the culture supernatant of dental pulp-derived stem cells preferably does not substantially contain serum (e.g., fetal bovine serum, human serum, sheep serum). Furthermore, it is preferable that the composition containing the culture supernatant of dental pulp-derived stem cells is substantially free of conventional serum substitutes such as Knockout serum replacement (KSR).The content (solid content) of each of the above-mentioned other components in the composition containing the culture supernatant of dental pulp-derived stem cells is preferably 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0044] <Method for preparing culture supernatant of dental pulp-derived stem cells> The culture supernatant of dental pulp-derived stem cells is not particularly limited. The culture supernatant of dental pulp-derived stem cells is preferably substantially free of serum. For example, the culture supernatant of dental pulp-derived stem cells preferably contains 1% by mass or less of serum, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0045] Dental pulp-derived stem cells may be derived from humans or non-human animals, including the same animals (biological species) as those to which the microparticles of the present invention are administered, as described below, and preferably mammals.

[0046] There are no particular limitations on the dental pulp-derived stem cells used in the culture supernatant. Stem cells from exfoliated deciduous teeth, deciduous tooth pulp stem cells obtained by other methods, and permanent tooth pulp stem cells (DPSCs) can be used. In addition to human deciduous tooth pulp stem cells and human permanent tooth pulp stem cells, dental pulp-derived stem cells derived from animals other than humans, such as porcine deciduous tooth pulp stem cells, can also be used. Dental pulp-derived stem cells can produce exosomes as well as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, and various cytokines such as chemokines. In addition, they can produce many other physiologically active substances. In the present invention, it is particularly preferable that the dental pulp-derived stem cells used in the dental pulp-derived stem cell culture supernatant are dental pulp-derived stem cells that contain many proteins, and it is preferable to use deciduous dental pulp stem cells. That is, in the present invention, it is preferable to use the deciduous dental pulp stem cell culture supernatant.

[0047] The dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as the desired treatment can be achieved. In particular, the present invention can use immortalized dental pulp-derived stem cells. By using immortalized stem cells that are capable of virtually unlimited proliferation, the amount and composition of biological factors contained in the stem cell culture supernatant can be stabilized over a long period of time. There are no particular limitations on the immortalized dental pulp-derived stem cells. It is preferable that the immortalized stem cells are non-cancerous immortalized stem cells. Immortalized dental pulp-derived stem cells can be prepared by adding the following low molecular weight compounds (inhibitors), either alone or in combination, to dental pulp-derived stem cells and culturing them. The TGFβ receptor inhibitor is not particularly limited as long as it has an effect of inhibiting the function of transforming growth factor (TGF) β receptor, and examples thereof include 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyridine, Examples of suitable ROCK inhibitors include pyrazole (A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all manufactured by Merck), and SB431542 (Sigma-Aldrich). A-83-01 is preferred. The ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-associated kinase. Examples of ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (all Fujifilm Wako Pure Chemical Industries, Ltd.). Preferred is Y-27632.The GSK3 inhibitor is not particularly limited as long as it inhibits GSK-3 (glycogen synthase kinase 3), and examples include A 1070722, BIO, and BIO-acetoxime (all manufactured by TOCRIS). The MEK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), and examples include AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86-9766), SL327, U0126-EtOH (all manufactured by Selleck), PD98059, U0124, U0125 (all manufactured by Cosmo Bio Co., Ltd.), and the like.

[0048] When the rejuvenating agent of the present invention is used in regenerative medicine, due to the requirements of the Act on Safety of Regenerative Medicine, the culture supernatant of dental pulp-derived stem cells or these immortalized stem cells, and compositions containing microparticles derived therefrom, are configured to contain no somatic stem cells other than dental pulp-derived stem cells. The culture supernatant of dental pulp-derived stem cells may contain mesenchymal stem cells or other somatic stem cells other than dental pulp-derived stem cells, but preferably does not contain them. Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermis, digestive system, bone marrow, nervous system, etc. Examples of somatic stem cells of the dermis include epithelial stem cells and hair follicle stem cells. Examples of somatic stem cells of the digestive system include pancreatic (general) stem cells and hepatic stem cells. Examples of somatic stem cells of the bone marrow system (other than mesenchymal stem cells) include hematopoietic stem cells. Examples of somatic stem cells of the nervous system include neural stem cells and retinal stem cells. The culture supernatant of dental pulp-derived stem cells may contain, but preferably does not contain, stem cells other than somatic stem cells. Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).

[0049] There are no particular limitations on the method for preparing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells, and conventional methods can be used. The culture supernatant of dental pulp-derived stem cells is a culture medium obtained by culturing dental pulp-derived stem cells. For example, a culture supernatant usable in the present invention can be obtained by separating and removing cellular components after culturing dental pulp-derived stem cells. Culture supernatants that have been appropriately subjected to various processes (e.g., centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) can also be used.

[0050] Dental pulp-derived stem cells for obtaining the conditioned medium can be selected by conventional methods based on cell size or morphology, or as adhesive cells. Adhesive cells or their subcultured cells can be selected from dental pulp cells collected from shed deciduous or permanent teeth. The conditioned medium for dental pulp-derived stem cells can be obtained by culturing selected stem cells.

[0051] The "dental pulp-derived stem cell culture supernatant" is preferably a culture medium that does not contain cells obtained by culturing dental pulp-derived stem cells. In one embodiment, the dental pulp-derived stem cell culture supernatant used in the present invention preferably does not contain cells (regardless of cell type) as a whole. This characteristic clearly distinguishes the composition of this embodiment from various compositions containing dental pulp-derived stem cells, as well as from dental pulp-derived stem cells themselves. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed solely of dental pulp-derived stem cell culture supernatant. The dental pulp-derived stem cell culture supernatant used in the present invention may contain both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells. The dental pulp-derived stem cell culture supernatant used in the present invention preferably contains deciduous dental pulp-derived stem cell culture supernatant as an active ingredient, more preferably at least 50% by weight, and particularly preferably at least 90% by weight. It is even more preferable that the dental pulp-derived stem cell culture supernatant used in the present invention be a composition composed solely of deciduous dental pulp-derived stem cell culture supernatant.

[0052] The culture medium for dental pulp-derived stem cells to obtain the culture supernatant can be a basal medium or a basal medium supplemented with serum, etc. Examples of basal media that can be used include Dulbecco's Modified Eagle's Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham's F12 Medium (HamF12) (Sigma, GIBCO, etc.), and RPMI 1640 medium. Examples of components that can be added to the medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various vitamins, and various minerals. However, to prepare a serum-free "dental pulp-derived stem cell culture supernatant," it is recommended to use a serum-free medium throughout the entire process or for the final or subsequent few subcultures. For example, serum-free culture supernatant of dental pulp-derived stem cells can be prepared by culturing dental pulp-derived stem cells in a serum-free medium. Serum-free culture supernatant of dental pulp-derived stem cells can also be obtained by performing one or more subcultures and culturing the last or penultimate subculture in a serum-free medium. Serum-free culture supernatant of dental pulp-derived stem cells can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or the like.

[0053] Conventional conditions can be applied to the cultivation of dental pulp-derived stem cells to obtain the culture supernatant. The method for preparing the culture supernatant of dental pulp-derived stem cells may be the same as the cell culture method described below, except that the stem cell isolation and selection steps are appropriately adjusted depending on the type of stem cells. Those skilled in the art can appropriately isolate and select dental pulp-derived stem cells depending on the type of stem cells. Furthermore, special conditions may be applied to the cultivation of dental pulp-derived stem cells in order to produce large amounts of microparticles such as exosomes. Examples of special conditions include low-temperature conditions, low-oxygen conditions, microgravity conditions, and conditions for co-culturing with some kind of stimuli.

[0054] The culture supernatant of dental pulp-derived stem cells used in the present invention for preparing microparticles such as exosomes may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but is preferably substantially free of other components. However, various additives used for preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.

[0055] (Preparation of Microparticles) Microparticles can be prepared by purifying them from the culture supernatant of dental pulp-derived stem cells or the like.

[0056] Purification of microparticles is preferably the separation of a fraction containing microparticles from the dental pulp-derived stem cell culture supernatant, and more preferably the isolation of microparticles. Microparticles can be isolated by separating them from non-associated components based on their properties. For example, microparticles can be isolated based on molecular weight, size, morphology, composition, or biological activity. Microparticles can be purified by centrifuging the dental pulp-derived stem cell culture supernatant and isolating a specific fraction (e.g., precipitate) rich in microparticles. Unnecessary components (insoluble components) from fractions other than the specified fraction may be removed. Complete removal of the solvent, dispersion medium, and unnecessary components from the microparticle composition is not required. Centrifugation conditions include 100 to 20,000 g for 1 to 30 minutes. Microparticles can be purified by filtering the dental pulp-derived stem cell culture supernatant or its centrifuged product. Unnecessary components can be removed by filtration. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed simultaneously. The material and pore size of the filtration membrane used for the filtration process are not particularly limited. Filtration can be performed using a known method using a filtration membrane with an appropriate molecular weight or size cutoff. From the perspective of easy exosome isolation, the pore size of the filtration membrane is preferably 10 to 1,000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. In the present invention, the culture supernatant of dental pulp-derived stem cells, its centrifuged product, or its filtrate can be separated using additional separation methods such as column chromatography. For example, high-performance liquid chromatography (HPLC) using various columns can be used. The column can be a size exclusion column or a binding column. One or more properties or biological activities of the microparticles can be used to track the microparticles (or their activity) in each fraction at each processing stage. For example, light scattering, refractive index, dynamic light scattering, or a UV-visible light detector can be used to track the microparticles. Alternatively, specific enzyme activity can be used to track the activity in each fraction.As a method for purifying microparticles, the method described in paragraphs

[0034] to

[0064] of JP-A No. 2019-524824 may be used, the contents of which are incorporated herein by reference.

[0057] The final form of the dental pulp-derived stem cell culture supernatant is not particularly limited. For example, the dental pulp-derived stem cell culture supernatant may be in the form of microparticles packed in a container together with a solvent or dispersion medium; microparticles gelled with a gel and packed in a container; or microparticles solidified by freezing and / or drying, formulated, or packed in a container. Examples of containers include tubes, centrifuge tubes, bags, and the like suitable for cryopreservation. The freezing temperature can be, for example, -20°C to -196°C.

[0058] Compared to compositions that can be used as conventional rejuvenation agents, the rejuvenation agent of the present invention has advantages such as ease of mass production, the ability to utilize stem cell culture medium that was previously discarded as industrial waste, and reduced disposal costs for stem cell culture medium. In particular, when the culture supernatant of dental pulp-derived stem cells is a culture supernatant of human dental pulp-derived stem cells, the rejuvenation agent of the present invention has the advantage of being highly safe from immunological and other standpoints and posing fewer ethical concerns when applied to humans. When the culture supernatant of dental pulp-derived stem cells is a culture supernatant of stem cells derived from the subject's own dental pulp, the rejuvenation agent of the present invention will be safer and pose fewer ethical concerns when applied to the subject. The rejuvenation agent of the present invention can also be used in restorative medicine. Compositions containing microparticles derived from the culture supernatant of dental pulp-derived stem cells are preferably used in restorative medicine. It is known that in regenerative medicine based on stem cell transplantation, stem cells are not the main players in regeneration, but rather the liquid components produced by stem cells, together with the subject's own stem cells, repair organs. The difficult problems associated with conventional stem cell transplantation, such as canceration, standardization, administration methods, storage, and culture methods, have been resolved, and restorative medicine is now possible using dental pulp-derived stem cell culture supernatant or compositions using microparticles derived therefrom. Compared to stem cell transplantation, the use of the rejuvenation agent of the present invention is safer, as tumorigenesis is less likely to occur because cells are not transplanted. Furthermore, the dental pulp-derived stem cell culture supernatant used in the rejuvenation agent of the present invention has the advantage of being of consistently standardized quality. Mass production and efficient administration methods can be selected, making it available at low cost.

[0059] [Rejuvenation Method] The rejuvenation method of the present invention comprises administering an effective amount of the rejuvenation agent of the present invention to a subject containing cells.

[0060] The process for administering the microparticles of the present invention to a subject is not particularly limited. Examples of administration methods include spraying or inhaling into the oral cavity, nasal cavity, or respiratory tract, infusion, topical administration, nasal drops, eye drops, and sublingual administration, with minimal invasiveness being preferred. Preferred local administration methods include injection, rectal administration (including catheters), and transurethral administration. Electroporation, which temporarily creates tiny holes in the cell membrane by applying a voltage (electric pulse) to the skin surface, allowing the active ingredient to penetrate into the dermis layer, which is difficult to reach with conventional care, is also preferred. Examples of local administration include intravenous administration, intraarterial administration, intraportal vein administration, intradermal administration, eye drops, sublingual administration, subcutaneous administration, transurethral administration, rectal administration, intramuscular administration, and intraperitoneal administration, with intraarterial, intravenous, subcutaneous, or intraperitoneal administration being more preferred. Furthermore, various formulation techniques can be used to alter the in vivo distribution of microparticles. Numerous methods for altering in vivo distribution are known to those skilled in the art. Examples of such methods include protecting exosomes in vesicles composed of substances such as proteins, lipids (e.g., liposomes), carbohydrates, or synthetic polymers. The rejuvenating agent of the present invention administered to a subject may circulate throughout the subject's body and reach the desired tissue. The number of administrations and the interval between administrations are not particularly limited. The number of administrations can be at least once per week, preferably at least five times, more preferably at least six times, and particularly preferably at least seven times. The administration interval is preferably between one hour and one week, more preferably between half a day and one week, and particularly preferably one day (once daily). However, this can be adjusted appropriately depending on the subject's species and symptoms. The rejuvenating agent of the present invention is preferably administered to a subject at least once per week over the effective therapeutic period. When the subject is a human, the number of administrations per week is preferably higher, with administration preferably being at least five times per week over the effective therapeutic period, and daily administration being preferred. In the present invention, the amount of the rejuvenating agent of the present invention administered is preferably an amount that results in 500 or more microparticles per cell, more preferably an amount that results in 800 or more microparticles per cell, and particularly preferably an amount that results in 1000 or more microparticles per cell.

[0061] There are no particular limitations on the cells (cell species) to which the rejuvenating agent of the present invention is administered. The cells to which the rejuvenating agent of the present invention is administered are preferably somatic cells, and more preferably somatic stem cells or differentiated cells. Examples of somatic stem cells include mesenchymal stem cells, neural stem cells, hematopoietic stem cells, vascular endothelial stem cells, hepatic stem cells, and epithelial stem cells, with mesenchymal stem cells being particularly preferred. Examples of mesenchymal stem cells include adipose-derived stem cells, umbilical cord-derived stem cells, dental pulp-derived stem cells, and bone marrow-derived stem cells. Examples of differentiated cells include cells other than stem cells and functional cells, specifically adipocytes. There are no particular limitations on the animals (biological species) to which the rejuvenating agent of the present invention is administered. The animals to which the rejuvenating agent of the present invention is administered are preferably mammals, birds (chickens, quail, ducks, etc.), and fish (salmon, trout, tuna, bonito, etc.). Mammals may be humans or non-human mammals, with humans being particularly preferred. More preferably, non-human mammals are cows, pigs, horses, goats, sheep, monkeys, dogs, cats, mice, rats, guinea pigs, and hamsters. The subject to which the rejuvenating agent of the present invention is administered may be the cells themselves. In this case, somatic cells are preferred, and somatic stem cells or differentiated cells are more preferred. The preferred ranges of somatic stem cells or differentiated cells are the same as the preferred ranges of somatic stem cells or differentiated cells described in the target cells (cell types) to which the rejuvenating agent of the present invention is administered.

[0062] The microparticles of the present invention may be used in combination with a conventionally known rejuvenating agent.

[0063] The features of the present invention will be explained in more detail below with reference to examples, comparative examples, and reference examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0064] Example 1 Preparation of Dental Pulp-Derived Stem Cell Culture Supernatant A culture supernatant of human deciduous dental pulp stem cells was prepared and separated according to the method described in Example 6 of Japanese Patent No. 6296622, except that DMEM medium was used instead of the DMEM / HamF12 mixed medium. Primary culture was performed with the addition of fetal bovine serum (FBS), and subculture was performed using the primary culture medium. The supernatant of the subculture medium was separated so as not to contain FBS, and the culture supernatant of deciduous dental pulp stem cells was prepared. Note that DMEM is Dulbecco's Modified Eagle's Medium, and F12 is Ham's F12 medium.

[0065] <Exosome Preparation> Dental pulp-derived stem cell exosomes were purified from the resulting dental pulp-derived stem cell culture supernatant using the following method. The culture supernatant (100 mL) of deciduous dental pulp stem cells was filtered through a 0.22-micrometer pore filter, and the resulting solution was centrifuged at 100,000 × g for 60 minutes at 4°C. The supernatant was decanted, and the exosome-enriched pellet was resuspended in phosphate-buffered saline (PBS). The resuspended sample was centrifuged at 100,000 × g for 60 minutes. The pellet was again collected from the bottom of the centrifuge tube as a concentrated sample (approximately 100 μl). Protein concentration was determined using a microBSA protein assay kit (Pierce, Rockford, IL). The exosome-containing composition (concentrated solution) was stored at -80°C. A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used as the rejuvenating agent (microparticle composition sample) of Example 1.

[0066] The average particle size and concentration of the microparticles contained in the rejuvenating agent of Example 1 were evaluated. The average particle size of the microparticles contained in the rejuvenating agent of Example 1 was 50 to 150 nm. The rejuvenating agent of Example 1 had a particle size of 1.0 × 10 ９ It is a highly concentrated exosome solution of 2.0 × 10 ９It was a highly concentrated exosome solution with exosome concentrations of 1000 / ml. Furthermore, the components of the obtained rejuvenating agent of Example 1 were analyzed by known methods. As a result, it was found that the rejuvenating agent of Example 1 did not contain stem cells derived from dental pulp, MCP-1, or Siglec-9. Therefore, it was found that the active ingredient of the rejuvenating agent of Example 1 was an active ingredient different from MCP-1 and Siglec-9, which are active ingredients in the culture supernatant of mesenchymal stem cells, and their analogs.

[0067] [Test Example 1]: (1) Cellular function recovery (search for microRNAs involved in rejuvenation) The small RNAs contained in the rejuvenating agent of Example 1 were analyzed by next-generation sequencing (NGS). NGS analysis identified 1,787 miRNAs contained in the rejuvenating agent of Example 1 (exosomes from dental pulp-derived stem cells). The miRNAs contained in the rejuvenating agent of Example 1 were searched for microRNAs involved in rejuvenation. IMOTA (Interactive Multi-Omics-Tissue Atlas) was used to extract miRNAs related to rejuvenation. IMOTA is an interactive multi-omics atlas that allows researchers to investigate the interactions and expression levels of miRNAs, mRNAs, and proteins in various tissues and cells (Nucleic Acids Research, Volume 46, Issue D1, January 4, 2018, Pages D770-D775, IMOTA: An interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions). Here, we explored whether rejuvenation-related miRNAs were included.

[0068] The microparticles contained in the rejuvenation agent of the present invention contained the following miRNAs as rejuvenation-related miRNAs: has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has-miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p, and miR-93-5p. Therefore, it was demonstrated that the rejuvenation agent of the present invention can restore cellular function.

[0069] The results of comparing the read counts of 50 miRNAs with the highest read counts among miRNAs expressed in exosomes from dental pulp-derived stem cells are shown in the graph in Figure 1. In Figure 1, the vertical axis represents the read count. In addition, the miRNAs enclosed in a box in Figure 1 are rejuvenation-related miRNAs.

[0070] 1, it was found that the microparticles contained in the rejuvenation agent of the present invention contain rejuvenation-related miRNA at a high concentration. Therefore, the rejuvenation agent of the present invention can restore cellular function.

[0071] Test Example 2: (2) Inhibition of Senescence-Associated Secretory Phenomenon (SASP) Using Cell Signaling Technology's Associated Secretary Phenotype (SASP) Antibody Sampler Kit #38461, SASP factors were quantitatively compared. Specifically, the SASP factors included in this kit are: IL-1β (D3U3E) Rabbit mAb #12703, CCL5 / RANTES (R40) Antibody #2987, CXCL10 (D5L5L) Rabbit mAb #14969, PAI-1 (D9C4) Rabbit mAb #11907, IL-6 (D3K2N) Rabbit mAb #12153, TNF-α (D5G9) Rabbit mAb #6945, and MMP-3 (D7F5B) Rabbit mAb #14351. The SASP factors were quantitatively compared using Carboxy-Terminal Antigen #39091. Anti-rabbit IgG and HRP-linked Antibody #7074 were used as secondary antibodies, and colorimetric quantification was performed.

[0072] Except for using umbilical cord-derived stem cells, a culture supernatant of umbilical cord-derived stem cells was prepared in the same manner as in Example 1. Except for using umbilical cord-derived stem cell culture supernatant, exosomes from umbilical cord-derived stem cells were purified in the same manner as in Example 1 (Comparative Example).

[0073] The rejuvenating agent of Example 1 (SHED-CM (deciduous tooth pulp stem cell culture supernatant)-EVs) was purified twice by ultracentrifugation, thawed in PBS(-), and stored in a deep freezer at -80°C for approximately 8 months. Umbilical cord-derived stem cell exosomes (umbilical cord MSC-EVs) were purified twice by ultracentrifugation, thawed in PBS(-), and stored in a deep freezer at -80°C for approximately 2 months. These EVs were treated at a ratio of 10,000 EVs / cell with senescent adipose mesenchymal stem cells at passage number P8, with a SA-β-gal positive cell count of 72%. After 72 hours, SASP factors were added according to the kit protocol. The results, shown as the average of N=3, are shown in Figure 2. FIG. 2 is a graph showing the expression of each SASP factor as a relative value (%), with Untreated being set at 100.

[0074] 2, it was found that the rejuvenating agent of the present invention can suppress senescence-associated secretory phenomenon (SASP). Specifically, it was found that it can suppress SASP factors such as IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3, and MCP-1. In particular, it was found that the rejuvenating agent of the present invention can significantly suppress CCL5, CXCL10, IL-6, TNF-α, and MCP-1 compared to exosomes from umbilical cord-derived stem cells.

[0075] [Test Example 3]: (3) Telomere Lengthening Using commercially available normal human dermal fibroblasts (NHDF cells), the average telomere length of chromosome ends was compared according to the passage number of NHDF cells. Telomere length was measured using a Cosmo Bio Telomere Length qPCR kit according to the protocol provided with the kit. The average telomere length of each chromosome end was calculated according to the following formula 1: Formula 1: (Average telomere length of each chromosome end) = (Total telomere length of target sample per diploid cell) / 92. First, for NHDF cells at passage number P5, the total telomere length of the target sample per diploid cell was 617 kB. According to formula 1, the average telomere length of each chromosome end was 617 / 92 = 6.70 kb. Next, NHDF cells at passage number P5 were plated into wells of a 24-well plate containing 1 mL of MSC growth medium. Cells in each well were stimulated at a ratio of 1,000 EVs / cell with 20 μL of PBS (Control) or the rejuvenating agent of Example 1 (culture supernatant of dental pulp-derived stem cells containing exosomes; shown in Figure 3 as SHED-CM-EVs) resuspended in 20 μL of PBS. The same procedure was then repeated each time passage was repeated, and in NHDF cells at passage number P12, the average telomere length at each chromosome end was 439 / 92 = 6.02 kb (SHED-CM-EVs). Meanwhile, in NHDF cells at passage number P12 (Control) that were passaged with the addition of PBS (Control), the total telomere length of the target sample per diploid cell was 439 kB, and according to Equation 1, the average telomere length at each chromosome end was 439 / 92 = 4.78 kb. The results are shown in Figure 3. From the above, it was found that the rejuvenating agent of the present invention can suppress the shortening of telomere length due to cell division compared to the control, and can lengthen telomeres compared to control cells of the same passage number.

[0076] [Test Example 4]: (4) Recovery of Cell Division Ability (Reduction in the Number of SA-β-gal Positive ADMSC Cells) Approximately 4,500 commercially available senescent ADMSCs (adipose-derived stem cells) at passage number P12 were plated into wells of a 24-well plate containing 1 mL of MSC growth medium. Cells in each well were stimulated with 20 μL of PBS (control) or the rejuvenating agent of Example 1 (a culture supernatant of dental pulp-derived stem cells containing exosomes; shown in Figure 4 as SHED-CM-EVs) resuspended in 20 μL of PBS at a ratio of 1,000 EVs / cell. After incubation for 24 hours (Day 1), 48 hours (Day 2), or 72 hours (Day 3), the cells were fixed and stained to determine SA-β-gal activity. A reference example was also performed in which cells in each well were stimulated with the rejuvenating agent of Example 1 at a ratio of 100 EVs / cell. Senescence-associated β-galactosidase (SA-β-gal) is specifically present in senescent cells. This hydrolase normally converts β-galactosides into monosaccharides and accumulates in lysosomes in senescent cells. In Test Example 4, senescent ADMSCs containing SA-β-gal were stained blue using X-GAL as a substrate using Cosmo Bio's Cellular Senescence Detection Kit (SA-β-Gal Staining) (product number: CBA-230) according to the protocol provided with the kit, and the number of SA-β-gal-positive cells was quantified. The number of SA-β-gal-positive cells was quantified as the average value of N = 3 independent cultures. The results are shown in Figure 4.

[0077] As shown in Figure 4, when the rejuvenating agent of Example 1 (exosome-containing dental pulp-derived stem cell culture supernatant, SHED-CM-EVs) was added at a concentration of 1,000 EVs / cell (1,000 particles of exosomes per cell), not only was the increase in the number of SA-β-gal-positive cells suppressed, but the number of SA-β-gal-positive cells was significantly reduced by 22.7% (Day 1), 29.9% (Day 2), and 31.6% (Day 3). This indicates that the rejuvenating agent of Example 1 can restore cell division ability. Figure 5 is a schematic diagram of Test Example 4, which demonstrates that the rejuvenating agent of the present invention (SHED-CM-EVs) has the effect of rejuvenating aged mesenchymal stem cells. Here, the relationship between the passage number of ADMSCs and the number of SA-β-gal-positive cells at 0 hours of culture time was measured in a separate test (N = 3). The results are shown in Figure 6. 6, the number of SA-β-gal positive cells in ADMSCs at 0 hours of culture was approximately 2% at passage number P2, approximately 9% at passage number P4, approximately 11% at passage number P6, approximately 15% at passage number P8, approximately 28% at passage number P10, and approximately 39% at passage number P12. Therefore, it can be said that the rejuvenating agent of Example 1 was able to rejuvenate P12 ADMSCs to the equivalent of P10. In other words, the rejuvenating agent of the present invention was able to suppress the number of SA-β-gal positive cells stained when administered to subjects passaged n = 12 times, compared to the number of SA-β-gal positive cells stained when administered to subjects passaged n = 12 times without administration of the rejuvenating agent.

[0078] From the above Test Examples 1 to 4, it was found that the rejuvenating agent of the present invention has all of the following effects: recovery of cell function, suppression of secretory phenomena associated with cellular aging, lengthening of telomeres, and recovery of cell division ability.

Claims

1. A rejuvenating agent for administration to a subject containing cells, the rejuvenating agent comprising microparticles derived from dental pulp-derived stem cells.

2. The rejuvenation agent according to claim 1, which is a composition comprising a culture supernatant of dental pulp-derived stem cells, the culture supernatant containing the microparticles.

3. The rejuvenation agent according to claim 1, wherein the microparticles (1) contain at least one of the following rejuvenation-related miRNAs: has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has-miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p and miR-93-5p.

4. The rejuvenation agent described in claim 3, wherein the microparticles contain at least one of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p as the rejuvenation-related miRNA.

5. The rejuvenation agent according to claim 3, wherein the microparticles contain the rejuvenation-related miRNA at a higher concentration than the culture supernatant.

6. The rejuvenating agent according to claim 1, wherein the composition does not contain the culture supernatant excluding the microparticles.

7. (2) The rejuvenating agent according to claim 1, which is an agent for suppressing the expression of the following SASP factors: IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3 and MCP-1.

8. The rejuvenation agent according to claim 1, wherein the cells are somatic stem cells or differentiated cells.

9. The rejuvenating agent described in claim 1, wherein the subject is a somatic stem cell or a differentiated cell, and when the rejuvenating agent is administered to a subject that has been passaged n times, where n is a natural number, the subject can be more rejuvenated than a subject that has been passaged n times without administration of the rejuvenating agent.

10. (3) A rejuvenation agent as described in claim 1, which inhibits shortening of the average telomere length at the ends of chromosomes possessed by the cells.

11. (4) A rejuvenating agent as described in claim 1, which suppresses the number of positive cells stained with SA-β-gal when administered to a subject that has been passaged n times compared to the number of positive cells stained with SA-β-gal in a subject that has been passaged n times without administration of the rejuvenating agent.

12. The rejuvenating agent according to claim 1, which has all of the following effects: recovery of cell function, inhibition of senescence-associated secretory phenomenon (SASP), lengthening of telomeres, and recovery of cell division ability.

13. The rejuvenating agent according to claim 1, which satisfies all of the following (1) to (4). (1) Rejuvenation-related miRNAs including at least one of the following rejuvenation-related miRNAs: has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has-miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p, and miR-93-5p; (2) An inhibitor of the expression of the following SASP factors: SASP factors: IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3, and MCP-1; (3) suppressing shortening of the average telomere length at the chromosome ends of the cells; (4) suppressing the number of positive cells stained with SA-β-gal in a subject that has been passaged n times when the rejuvenating agent is administered to the subject, compared to the number of positive cells stained with SA-β-gal in a subject that has been passaged n times without administration of the rejuvenating agent.

14. The rejuvenation agent according to claim 1, wherein the microparticles are exosomes.

15. A method of rejuvenation comprising administering an effective amount of the rejuvenation agent of claim 1 to a subject containing cells.

16. The rejuvenation method according to claim 15, wherein the dosage of the rejuvenation agent is an amount resulting in 500 or more microparticles per cell.

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